Naturally occurring antibodies (immunoglobulins) comprise two heavy chains linked together by disulfide bonds and two light chains, each light chain being linked to one of the heavy chains by disulfide bonds. Each chain has an N-terminal variable domain (VH or VL) and a constant domain at its C-terminus; the constant domain of the light chain is aligned with and disulfide bonded to the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. The heavy chain constant region includes (in the N- to C-terminal direction) the CH1 and hinge regions. The light chain also contains a hinge domain. Particular amino acid residues are believed to form an interface between and disulfide bond the light and heavy chain variable domains, see e.g. Chothia et al., J. Mol. Biol. 186:651-663 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82:4592-4596 (1985); Padlan et al., Mol. Immunol., 23(9): 951-960 (1986); and S. Miller, J. Mol. Biol., 216: 965-973 (1990).
The constant domains are not involved directly in binding the antibody to an antigen, but are involved in various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity and complement dependent cytotoxicity. The variable domains of each pair of light and heavy chains are involved directly in binding the antibody to the antigen. The domains of natural light and heavy chains have the same general structure, the so-called immunoglobulin fold, and each domain comprises four framework (FR) regions, whose sequences are somewhat conserved, connected by three hyper-variable or complementarity determining regions (CDRs) (see Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). The four framework regions largely adopt a β-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site.
Antibodies can be divided into a variety of antigen-binding fragments. The Fv fragment is a heterodimer containing only the variable domains of the heavy chain and the light chain. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the designation herein for Fab′ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which are between hinge cysteines.
Recombinant production of antibodies and antibody fragments facilitates the engineering of antibodies with enhanced antigen binding affinities, modified immunogenicity, and also of bifunctional antibodies. The first expression systems reported with which functional antibodies could obtained were for mammalian cells. The U.S. patent of Cabilly et al., U.S. Pat. No. 4,816,567, for example, teaches a method of co-expressing at least the variable region sequences of light and heavy chains in host cells. Other researchers in the field have reported baculovirus expression systems (Haseman et al., Proc. Natl. Acad. Sci. USA 87:3942-3946 (1990), yeast systems (Horwitz et al., Proc. Natl. Acad. Sci. USA, 85:8678-8682 (1988), combinatorial libraries in phage lambda (Huse et al., Science 246:1275-1281 (1989), and work with filamentous phage (McCafferty et al., Nature 348:552-554 (1990).
The production of antibodies and antibody fragments in bacterial systems have been pursued by workers in the field, particularly in E. coli expression systems. There are several advantages to E. coli expression systems, including a well-studied and convenient gene technology which permits constructs to be made easily and directly expressed, and the relatively convenient and economical large-scale production of product made possible by the fast growth of E. coli and its comparatively simple fermentation. The large-scale production of functional antibody fragments in E. coli would be valuable for research as well as commercial applications.
The expression of antibody genes in bacteria was reported by Cabilly et al., Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984), Boss et al., Nucleic Acids Res. 12:3791-3806 (1984); these reports show cytoplasmic expression and rather variable yields were reported. Zemel-Oreasen et al., Gene 315-322 (1984) report the secretion and processing of an immunoglobulin light chain in E. coli. Plückthun et al., Cold Spring Harbor Symposia on Quantitative Biology, Volume LII, pages 105-112 (1987, Cold Spring Harbor Laboratory) disclose expression of a cytoplasmic hybrid protein, a potentially exportable hybrid protein, and expression and periplasmic transport of VL, VH, VLCL, and VHCH chains as fusions with an alkaline phosphatase or β-lactamase signal sequences. Skerra and Plückthun, Science 240:1038-1041 (1988) report the periplasmic secretion and correct folding in vivo of the variable domains of an antibody to the E. coli periplasm; a similar strategy and results were reported by Better et al., Science 240:1041-1043 (1988) for expression of a murine Fab fragment.
Bird et al., Nature 332:323-327 (1989) report the linkage of the light and heavy chain fragment of the Fv region via an amino acid sequence, and production of the complex as a single polypeptide in E. coli; see also Ladner et al., U.S. Pat. No. 4,946,778. Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988) report similar work. Ward et al., Nature 341:544-546 (1989) report the production in and secretion of “single-domain” antibodies (isolated heavy chain variable domains) from E. coli. Condra et al., Journal of Biological Chemistry, 265(4):2292-2295 (1990) disclose the expression of cDNAs encoding antibody light and heavy chains in E. coli and their renaturation into Fab fragments. Better and Horwitz, Methods in Enzymology, 178:476-496 (1989), describe the expression, and secretion of functional Fab fragments from E. coli and Saccharomyces cerevisiae. 
Plückthun and Skerra describe techniques for the expression of functional antibody Fv and Fab fragments in E. coli in Methods in Enzymology 178:497-515 (1989). According to their strategy, in the cytoplasm, the precursor proteins for VL and VH, each fused to a bacterial signal sequence, are synthesized in reduced form. After translocation through the inner membrane into the periplasm, the signal sequences are cleaved, the domains fold and assemble, and the disulfide bonds form. They teach that expression of the Fab fragment according to their strategy is analogous. Similar expression strategies are found elsewhere in the literature. See also Plückthun, Biotechnology, 9:545-551 (1991) for a review of E. coli expression of antibody fragments.
Cabilly (Gene, 85:553-557 [1989]) teaches that, in E. coli cells growing at reduced temperatures (21° C. or 30° C., rather than at 37° C.), a single expression plasmid coding for kappa-chains and truncated heavy chains (Fd fragments) gives rise to high yields of functional Fab fragments. Cabilly discusses that the Fab fragments seem to exist in the E. coli cytoplasm as non-covalently linked dimers, but that soluble Fab fragments isolated from E. coli appear as covalent dimers, formed by air oxidation following cell rupture.
It is known in the literature that in the presence of low concentrations of a mild reductant such as cysteamine the bivalent F(ab′)2 antibody fragment dissociates into two Fab′ fragments. This dissociation is reversible by mild oxidation. The production of Fab and F(ab′)2 antibody fragments has also been shown by partial reduction and limited proteolysis of intact antibodies, see e.g. Parham, in Cellular Immunology (E. M. Weir, Ed., Blackwell Scientific, CA) 4th edition, vol. 1 chapter 14 (1983), however with these methods it is difficult to control the precise nature and proportions of the antibody fragment recovered. Bivalent antibodies are those which contain at least two epitopic combining sites (which sites may be on the same or different antigens).
Bispecific antibodies are bivalent antibodies capable of binding two epitopes not shared by a single antigen. Bispecific monoclonal antibodies (BsMAbs) with dual specificities for tumor-associated antigens on tumor cells and for surface markers in immune effector cells have been described (see, e.g. Liu et al., Proc. Natl. Acad. Sci. USA 82:8648 (1985): Perez et al., Nature 316:354 (1985)). These BsMAbs have been shown to be effective in directing and triggering effector cells to kill tumor cell targets (Fanger et al., Immunol. Today 12:51 (1991)). One approach to the production of BsMabs involves the fusion of two monoclonal antibody-producing hybridomas to form quadromas (hybrid hybridomas) which secrete BsMab in addition to undesirable chain combinations including parental MAbs (Milstein, C. and Cuello, A. C., Nature, 305:537 [1983]). However, for production of bispecific humanized antibodies and antibody fragments, other techniques would be preferred.
Nisonoff and Mandy (Nature 4826:355-359 (1962)) describe the digestion of rabbit antibodies and subsequent recombination of the antibody fragments; they disclose that antibody molecules of dual specificity can be obtained by combining univalent fragments of pepsin-treated antibodies of different specificities. See also Hammerling et al., Journal of Experimental Medicine 128:1461-1469 (1968); Parham, Human Immunology 12:213-331 (1985); Raso and Griffin, Cancer Research 41:2073-2076 (1981); and Paulus (U.S. Pat. No. 4,444,878).
Another approach utilizes directed chemical coupling of bispecific Fab′ fragments from two different MAbs to assemble a BsMAb, in this case a F(ab′)2, with the desired specificities (see e.g., Nolan et al., Biochimica et Biophysica Acta 1040:1 (1990). See also R. A. Maurer's Ph.D. Thesis, Harvard University (1978), and Brennan et al., Science 229:81-83 (1985) for chemistries for the directed coupling of dithionitrobenzoate derivatives of Fab′ fragments. Brennan et al. also teach the use of use sodium arsenite to cross-link two proximate cysteines, however this reaction involves highly toxic compounds. (Glennie et al., J. Biol. Chem., 141(10): 3662-3670 [1985] and J. Immunol., 139:2367-2375 [1975]) teach the preparation of bispecific F(ab′)2 antibody fragments containing thioether linkages. These chemistries would also be applicable for the coupling of identical Fab′ fragments.
Lyons et al., Protein Engineering 3(8)703-708 (1990) teach the introduction of a cysteine into an antibody (there the CH1 domain of a heavy chain) and the site-specific attachment of effector or reporter molecules through the introduced cysteine.
Despite the advances in E. coli expression of functional antibody fragments shown in the literature, there remains a need for efficient and economical techniques for the production of bivalent antibodies, particularly F(ab′)2 molecules, and for methods which permit the tailoring of bivalent and bispecific F(ab′)2 molecules. It would be desirable to produce stable Fab′-SH polypeptides which may be conveniently coupled in vitro to form bivalent Fv or F(ab′)2 molecules.
It is therefore an object of this invention to provide methods for the preparation of polypeptides comprising Fv domains, particularly Fab′, Fab′-SH and F(ab′)2 antibody fragments, in or derived from bacterial cell culture in high yield.
It is a further object of this invention to provide methods for the efficient preparation of homogenous bivalent and bispecific F(ab′)2 antibody fragments.
It is another object of this invention to provide Fab′ antibody fragments having at least one hinge region cysteine present as a free thiol (Fab′-SH). It is a related object to obviate the inherent problems in generating Fab′-SH from intact antibodies: differences in susceptibility to proteolysis and non-specific cleavage, low yield, as well as partial reduction which is not completely selective for the hinge disulfide bond(s). It is another object of the present invention to prevent intra-hinge disulfide bond formation without resorting to the use of highly toxic arsenite to chelate vicinal thiols, or other inefficient and undesirable methods.
Other objects, features, and characteristics of the present invention will become apparent upon consideration of the following description and the appended claims.